There has been rapidly increasing interest in reprogramming, since a study indicating the production of induced pluripotent stem cells from human fibroblasts was reported in 2007. Human embryonic stem cells used in previous stem cell studies have problems in that they pose ethical issues by the use of human embryos, cause immune rejection, and form a teratoma when undifferentiated embryonic stem cells are transplanted. Furthermore, adult stem cells have problems in that they are difficult to obtain and the differentiation potential thereof is limited. However, induced pluripotent stem cells avoid ethical issues and have no immune rejection, but may cause problems associated with teratoma formation when undifferentiated stem cells are transplanted. Although induced pluripotent stem cells have properties similar to those of embryonic stem cells, viral systems that are mainly used to form induced pluripotent stem cells may cause mutations by random integration of genes. In order to overcome the problem of viral systems, plasmids, proteins, RNAs or the like are used, but they have low efficiency, and may cause new problems due to the use of oncogenes.
In attempts to overcome the problems of such induced pluripotent stem cells, studies on direct conversion of human fibroblasts into desired cells using direct conversion method have been reported. Among them, direct conversion into neural cells using fibroblasts for the treatment of intractable brain diseases has been actively conducted, and the study for generation of neural cells were successful through various combination of neuron-related transcription factors have been introduced into human fibroblasts. These studies demonstrated their potential for use as cellular therapeutic agents against intractable brain diseases, but there was difficulty in obtaining neural cells in amounts sufficient for use in cell therapy, because neural cells are already differentiated cells.
Due to this problem, in recent years, methods for allowing fibroblasts to directly differentiate into neural stem cells have been studied. Most methods induce neural stem cells from fibroblasts by introducing various transcription factors using the viral system, however, in more recent, it has reached a level that can induce neural stem cells using only single transcription factor. Neural stem cells are self-renewable, and thus can be obtained in desired amounts, and are capable of differentiating into neural cells. Furthermore, neural stem cells derived from fibroblasts of each individual by direct conversion pose no ethical issues, have no immune rejection, and do not induce tumorigenesis after transplantation. Thus, induced neural stem cells are very useful as cellular therapeutic agents against intractable brain diseases.
Cell therapy is a therapeutic method that transplants experimentally derived healthy cells to replace damaged cells and tissues in the human body. In the case of patients having diseases caused by genetic factors, skin cells may be directly converted into desired cells, and the desired cells may be transplanted after abnormal genes therein are replaced with normal genes by genetic manipulation. However, because direct conversion methods that have been studied before are methods that introduce foreign genes, cells obtained by these methods are difficult to actually use as cellular therapeutic agents.
Accordingly, the present inventors have made extensive efforts to induce neural stem cells from fibroblasts using small-molecule compounds without any introduction of a foreign gene, and as a result, have found that neural stem cells can be produced which are capable of proliferating in a sufficient amount required for transplantation and which are genetically stable (genomic DNA stability) without inducing tumorigenesis, thereby completing the present invention.